Nanoantibodies, Binding Chlamydia Trachomatis Antigen, Method for Inhibition of Infection Induced by Chlamydia Trachomatis

ABSTRACT

A nanoantibody specifically binding surface antigen of  Chlamydia trachomatis  and having SEQ ID NO:2 amino acid sequence is disclosed. A nanoantibody specifically binding surface antigen of  Chlamydia trachomatis  and having SEQ ID NO:4 amino acid sequence is disclosed. A nanoantibody with SEQ ID NO:2 amino acid sequence inhibits development of  Chlamydia  infection caused by  C. trachomatis . A nanoantibody with SEQ ID NO:4 amino acid sequence inhibits development of  Chlamydia  infection caused by  C. trachomatis . A method of in vitro inhibiting a  Chlamydia  infection caused by  C. trachomatis  has the steps of pretreating elementary bodies of  C. trachomatis  by a therapeutically efficient amount of a nanoantibody specifically binding to a surface antigen of  Chlamydia trachomatis , the nanoantibody comprising an amino acid sequence SEQ ID NO: 4 or SEQ ID NO:4, and then adding the elementary bodies of  C. trachomatis  to target cells being infected.

RELATED APPLICATIONS

This application is a Continuation application of InternationalApplication PCT/RU2013/000227, filed on Mar. 19, 2013, which in turnclaims priority to Russian Patent Applications No. RU 2012101955, filedJan. 20, 2012, both of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The invention refers to biotechnology and medicine, especially toisolation and use of single domain nanoantibodies for detection andsuppression of infection induced by pathogenic microorganisms,especially, Chlamydia trachomatis.

BACKGROUND OF THE INVENTION

C. trachomatis is a gram-negative bacterium that refers to an obligateintracellular human pathogen. Chlamydia has two-phase cycle ofdevelopment that consists of two forms of pathogen existence:extracellular elementary bodies and intracellular reticular bodies.

One of the most common bacterial STD in men and women is an urogenitalinfection (UGI) caused by C. trachomatis.

There are over 20 nosological forms among diseases induced by C.trachomatis species where special role plays sexually transmittedurogenital Chlamydia infection (UGCI) affecting human urogenital tract.Urogenital Chlamydia infection—cervicitis, urethritis, proctitis,endometritis, salpingitis, perihepatitis—in women; urethritis,epididymitis, proctitis, prostatitis—in men—are most common amongsexually active population.

Chlamydia are detected in 50-57% of sterility cases. There are observednot only functional disorders of reproduction, but also involvement ofhomeostasis control systems, immune competent cells etc. Chlamydiainfections incidence in case of tubal infertility is from 41 to 54%.After first case of chlamydiosis the risk of tubal infertility increasesby 10%, after third case—by 50%. In infertile couples 50-55% of men aresterile, and in 64% of these cases sterility is caused by UGCI.Chlamydia can cause infertility as a result of direct exposure on spermdue to tight adhesion of Chlamydia on male gametes that preventsimpregnating the ovum. Some authors note that secondary femaleinfertility in case of ascending UGCI is observed more often (in 6times) than in case of gonorrhea.

C. trachomatis is observed in 9-30% of cases of ectopic pregnancy. Inrecent years has been noted increase of chlamydiosis of pregnant(10-40%) and newborns. In 40-60% of cases infected women transmitinfection to newborns.

UGI's are diagnosed in 46% of cases at the age of 15-19 years old, andin 30% at the age of 20-24 years old. Incidence is quite high not onlyamong adult population and teenagers having sexual relations but alsoamong younger children without sexual relations. Thus, C. trachomatishas been observed in 67.4% of boys under 12 years old with UGI. In thiscase clinical picture of UGCI conformed to urethritis, andultrasonography showed traces of past prostatitis in 7.9% of children.

It is very important that 75% of women and 40% of men had asymptomaticdisease, and 30-40% of teenagers had latent Chlamydia infectionproceeding from 2 to 5 years. Asymptomatic disease is typical not onlyfor Urogenital localization, but also for infections of other organs.Epidemiological importance of asymptomatic Chlamydia infection was shownin works of A. Shatkin, the founder of Chlamydiology in Russia.Chlamydia isn't a member of normal human flora. Detection of itindicates infectious process, and the absence of clinical symptomsdetermines only temporary balance between a parasite and a host underconditions limiting but not inhibiting growth of intracellularpathogens. In this regard, Chlamydia infection with clinicalasymptomatic course is dangerous as its manifest forms and needstreatment and prevention. Undiagnosed, untreated or improperly treatedpatients with acute, subacute or slow Chlamydia inflammatory processesare most attributable to incidence of infection nowadays.

In many developed countries of Europe and the US national UGCI controlprograms based on screening of high-risk population, early treatment,examination and treatment of partners have been realized for over 25years. Despite these actions, cases of primary chlamydiosis andreinfection have still increased.

During recent years incidence of urogenital Chlamydia infections in theRussian Federation has come out on top among all the sexuallytransmitted bacterial infections and it gives way only totrichomoniasis.

According to Central Scientific and Research Institute for Organizationand Information Support of Health Care FSU, UGCI incidence comparing to1994 increased in 1.7 times (61.4 versus 101.7). In 2005, total numberof STD cases amounted to 503.6 per 100000 of population, in2006—decreased to 4.1%. However, UGCI in 2005 amounted to 95.9 per100000 (adults) and 3.1 per 100000 (children); in 2006—97.2 per 100000;in 2007—91.1 per 100000 (adults) and 3.2 per 100000 (children).

In 2008, 611634 cases of sexually transmitted infections wereregistered, that amounted to 403.5 per 100000 of population. Chlamydiainfection amounted to 20.8%. For the last 3 years reduction of patientswith STD (including 8.4% with chlamydia infection) is observed all overthe Russia. Incidence of Chlamydia infection in Russia in 2008 was 89.5per 1000000 of adult population and 2.8 per 100000 of children.

So medical and social role of Chlamydia infection is firstly caused byhigh incidence and frequent complications, and also by effect ondemographic determinants due to fact that UGCI is the most frequentcause of female and male infertility.

Urogenital chlamydiosis therapy represents the most difficult aspect ofthe concerned issue, which is associated not only with features ofinfectious agent and its development cycle but also with association ofchlamydiosis with other infection in 70% of cases: ureaplasma, herpessimplex virus, Gardnerella vaginalis etc. Causal treatment is based onchlamydial antibiotics susceptibility. Widespread use of tetracyclineantibiotics in treatment of chlamydiosis (doxycycline, metacycline,minocycline, and tetracycline) is quite reasonable. According to WHOrecommendations: 100 mg doxycycline orally twice a day or 500 mgtetracycline orally 4 times a day, course of treatment—7 days. It mustbe noted that number of ineffective treatment cases has significantlyincreased recently, which, probably, may be due to resistance ofcausative agent to antibiotic in population. Doses and treatmentduration must be adjusted evidently depending on UGCI course (acute,chronic, ascending infection, exacerbation etc.). The most conflicts ofresearchers are related to use of fluroquinolone antibiotics: ofloxacin,pefloxacin, ciprofloxacin. Some authors report efficient ofloxacintherapy in 81-100% of cases (200-300 mg per os twice a day during 7days); others report high failure rate and the worst long-term outcomes.Treatment of chronic, complicated forms of chlamydiosis currentlypresents very serious and unresolved problem. This is firstly related tofact that during chronization of infectious process in macroorganismoccur persisting forms of Chlamydia that are antibiotic-resistant andare adjusted to long term survival. That is why treatment of chronicforms of UGCI with antibiotics, according to numerous clinical andmicrobiological studies, is inefficient. The situation makes developmentof new antibacterial products, mechanism of action of which shall befundamentally different comparing to antibiotic action, very important.

Murine monoclonal antibodies specific to major outer membrane protein(MOMP) of C. trachomatis, can act as the closest technical decisionanalog, making the ground of the present invention. Antibodies wereprepared by standard method of isolation of monoclonal antibodies basedon hybridome technology with mice immunization. Isolated antibodiesrecognized MOMP of C. trachomatis, identifying epitopes, localized atthe surface of Chlamydia cell. Antibodies reduced toxicity of causativeagent in mice in vivo.

Zhang, Y.-X., S. J. Stewart, and H. D. Caldwell. Protective monoclonalantibodies to Chlamydia trachomatis serovar- and serogroup-specificmajor outer membrane protein determinants. Infect. Immun. 1989,57:636-638 (SUPPLEMENT 1)

This technical decision as the closest to the claimed one regardingactive ingredient composition and mode of its use has been chosen by theauthors of this invention as a prototype.

The disadvantages of the prototype are:

1) Relatively expensive production of antibodies, difficulties inmaintaining and storage of the producer, extremely high requirements toquality of the used reagents and culture conditions.

2) Relatively large size of isolated antibodies resulting in low tissuepermeability.

3) Structural characteristics impose restrictions on recognition of some“hidden” epitopes located, in clefts, fissures of small size in proteinstructures.

4) Limitation and relative complexity of genetic engineeringmanipulations, adaptations for specific issues, difficulties in creationof multivalent and multifunctional derivatives of specified antibodies.

Thus, there is a need in development of newantibodies—antigen-recognizing molecules without any of the saiddisadvantages and specifically recognizing C. trachomatis, in thetechnical level.

SUMMARY OF THE INVENTION

The object of present invention is a creation of new antibodies, able toeffectively recognize antigens of C. trachomatis and inhibit chlamydialinfection. Its isolation, production and storage must be cost-effective,efficient and about simple. It must be much smaller than classicalantibodies.

Assigned problem is solved by construction of nanoantibody (with SEQ IDNO:2 amino acid sequence) specifically binding to surface Chlamydiatrachomatis antigen. Nanoantibody, specifically binding surface antigenof Chlamydia trachomatis, with SEQ ID NO:4 amino acid sequence has alsobeen constructed. Nanoantibody with SEQ ID NO:2 amino acid sequenceinhibits development of Chlamydia infection induced by C. trachomatis.Nanoantibody with SEQ ID NO:4 amino acid sequence inhibits developmentof Chlamydia infection induced by C. trachomatis. Method of inhibitionof Chlamydia infection in vitro, induced by C. trachomatis, involvingpretreatment of elementary bodies of C. trachomatis by therapeuticallyefficient volume of nanoantibody with SEQ ID NO:2 amino acid sequencebefore adding it to target cell, has been claimed. Method of inhibitionof Chlamydia infection in vitro, induced by C. trachomatis, involvingpretreatment of elementary bodies of C. trachomatis by therapeuticallyefficient volume of nanoantibody with SEQ ID NO:4 amino acid sequencebefore adding it to target cell, has been claimed.

The basis of the invention are not classical bivalent antibodies,considered as a prototype, but small nanoantibodies with variety ofadvantages comparing to classical monoclonal antibodies for practicaluse in the sphere of disease therapy. As long as nanoantibodies withmolecular mass of about 12-15 kDa are 10 times smaller than the size oftraditional antibodies, they get numerous positive features of practicalimportance. There are efficient ways of isolation and selection of suchantibodies specific to various antigens and, due to their lowimmunogenicity, nanoantibodies may be used for treatment of infectionsinduced by pathogens of this family.

Absolute equivalent of the term “nanoantibodies” for the purposes of thepresent invention is a widely used denomination “NANOBODY”, introducedby ABLYNX, and also “single-domain mini-antibody” and “single-domainnanoantibody”.

Recombinant nanoantibodies production is based on specific nonclassicsingle-chain antibodies, existing naturally together with classicantibodies in Camelids (and some species of cartilaginous fishes). Thesespecific antibodies consist of dimer of only one short (without firstconstant CH1 region) heavy immunoglobulin chain and are fully functionalin the absence of the light immunoglobulin chain. Only one variabledomain (VHH, “nanoantibody”, “nanobody” or single-domain nanoantibody)of this antibody is necessary and sufficient for specific recognitionand binding of antigen. Organization of variable domains (VHH) ofnonclassic antibodies is largely similar to that of the variable domains(VH) of classic antibodies (human VH-domains of subclass IgG3immunoglobulins have most evident homology with VH and VHH of Camelids).In both cases V-domains consist of four conservative framework regions(FR), surrounding three hypervariable complementarity determiningregions CDR. In both cases domains form typical for immunoglobulinV-domain spatial structure of two beta-layers: the first—from four aminoacid chains, and the second—from five [Padlan E. A. X-Raycrystallography of antibodies. Adv. Protein Chem. 1996; 49: 57-133.Muyldermans S., Cambillau C., Wyns L. Recognition of antigens bysingle-domain antibody fragments: the superfluous luxury of paireddomains. TIBS 2001; 26: 230-235]. In this structure all of threehypervariable regions cluster at one side of V-domain (where they takepart in recognition of antigen) and are located in loops bindingbeta-structures. However, there are also significant distinctions,attributed to VHH functioning in the single domain format. Thus,hypervariable CDR1 and CDR3 regions are visibly increased in case ofVHH. Cysteine residues are often detected in hypervariable VHH regionsbeing present in two regions at one time (the most often in CDR1 andCDR3, less often—in CDR2 and CDR3). It was shown that these cysteineresidues formed disulfide bonds that leaded to additional stabilizationof the present antigen loop structure in crystal structures analysis ofVHH. The most obvious and reproductive distinctive feature of VHH arefour changes of hydrophobic amino acid residues to hydrophilic in thesecond framework region (Val37Phe, Gly44Glu, Leu45Arg, Trp47Gly,according to Kabat Numbering Scheme). This framework region in case ofVH domain is highly conserved, enriched with hydrophobic amino acidresidues, and is critical for bonding with VL variable domain of thelight chain. VHH-domain is quite different in this context: thedesignated changes of hydrophobic amino acids to hydrophilic let toimpossibility of VHH and VL association. Such changes also explain highsolubility of VHH, nanoantibody, when it is isolated in the form ofrecombinant protein [Tillib S. V. “camel antibodies”—effectiveinstrument for studies, diagnostics and therapy. Molecular biology 2011;45(1): 77-85].

Camel nanoantibodies have variety of advantages which allows to assumegreat potential for their future use in various studies and in creatingnew biotechnological devices, and also for clinical purposes indiagnostics and treatment of diseases compared to traditional and purelyrecombinant antibodies.

Unique features of nanoantibodies determining great potential of theiruse for variety of practical applications in immune biotechnology are[See overview: Tillib S. V. “camel antibodies”—effective instrument forstudies, diagnostics and therapy. Molecular biology 2011; 45(1): 77-85];

1) Highly efficient method of generating and selection ofnanoantibodies.

2) Small size, ˜2×4 nm, 13-15 kDa, enhanced cell permeability.

3) Structural properties, i.e. ability to form unusual for classicalantibodies paratopes, allowing to bind with clefts and binding sites ofproteins; can be used for detection of “hidden” epitopes that cannot berecognized by ordinary antibodies.

4) High expression rate, cost efficient development in large volume.Nanoantibodies are usually developed in E. coli periplasm (amounting to1-10 mg of 1 L of culture). Possible development in yeast, plants andcells of mammals.

5) Simplicity of all the possible genetic engineering manipulations,adaptations for specific issues, possibility to create multivalent andmultifunctional derivatives.

6) Low immunogenicity; possibility to economically “humanize” antibodieswithout significant loss of their specific activity.

Possibility to isolate recombinant nanoantibodies with given specificityis determined by functional nonclassic antibodies with quite widerecognition spectrum intrinsic to representatives of Camelidae family.Nonclassic antibodies consist of dimer of only one short heavyimmunoglobulin chain without light chains, recognition specificity ofwhich is determined by only one variable domain [Hamers-Casterman C,Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoidof light chains. Nature 1993; 363:446-448]. Technical realization ofselection of nanoantibodies (that are genetically engineered derivativesof antigen-recognizing domains of single chain camel antibodies) isbased on high-efficient selection procedure of antigen-recognizingpolypeptides, exposed on the surface of the filamentous phageparticle—“phage display”.

Phage display method is quite efficient and widely used technique forfunctional selection of DNA sequences from large recombinant libraries,encoding peptides and proteins, having given properties and expressed onthe surface protein composition of filamentous phages [Brissette R &Goldstein N I. The use of phage display peptide libraries for basic andtranslational research. Methods Mol Biol. 2007; 383:203-13; Sidhu S S &Koide S. Phage display for engineering and analyzing protein interactioninterfaces. Curr Opin Struct Biol. 2007; 17:481-7]. One of the mostimportant applications of this technique is generation of specificrecombinant antibodies for different antigens [Hoogenboom H R. Selectingand screening recombinant antibody libraries. Nat Biotechnol. 2005;23:1105-16]. Normally hybrid recombinant single-chain proteins are usedinstead of large whole molecules of classic antibodies for exposure atthe phage surface. These hybrid recombinant single-chain proteins haverandom combinations of cloned sequences in variable regions of heavy andlight immunoglobulin chains bound with short serine/glycine-enrichedlinker sequence. Such chimeric molecule in case of right domaincombination can keep specificity of initial immunoglobulin, despite ofthe implemented changes compared to native antibodies molecule. One ofthe problem in traditional recombinant technologies is need to work withextremely large libraries of recombinant antibodies, where all possiblecombinations of two random variable regions (heavy and light chains ofimmunoglobulins) bound by linker sequence must be represented. There isalso another problem apart from the representation issue. This problemconsists of formation of the right relative conformation of these twodomains and also of solubility of individual variable domains very oftentending to adhesion. These issues can be avoided using nanoantibodies,as long as virtually every cloned variable domain of single-chainantibodies shall in this case possess a certain antigen-recognizingspecificity, corresponding to one of the antibodies of immunized animal,and selection can be effectively performed from relatively smalllibraries of such domains.

Nanoantibodies with certain specificity or their derivatives can beused, as can classical antibodies, in various applications, including,but not limited to, antigen detection both for research and diagnosticpurposes, suppression of protein-antigen activity, specific delivery bybinding to antigen of desired molecules, conjugated with antibody.Nanoantibodies can also be initial modules-units of more complicatedmultimodule products. Unification in one multivalent derivative of two,three and more monovalent primary nanoantibodies is possible. Thesenanoantibodies unified into one construct can be bound to the sameepitope of target antigen, and its different epitopes, or even tovarious target antigens. Combined unification into one construct ofnanoantibodies and other molecules or products for obtainment ofmultifunctional products is also possible [Conrath K E, Lauwereys M,Wyns L, Muyldermans S. Camel single-domain antibodies as modularbuilding units in bispecific and bivalent antibody constructs. J BiolChem. 2001 Mar. 9; 276 (10): 7346-50; Zhang J, Tanha J, Hirama T, KhieuN H, To R, Tong-Sevinc H, Stone E, Brisson J R, MacKenzie C R.Pentamerization of single-domain antibodies from phage libraries: anovel strategy for the rapid generation of high-avidity antibodyreagents. J Mol Biol. 2004 Jan. 2; 335 (1): 49-56; Cortez-Retamozo V,Backmann N, Senter P D, Wernery U, De Baetselier P, Muyldermans S,Revets H. Efficient cancer therapy with a nanobody-based conjugateCancer Res. 2004 Apr. 15; 64 (8): 2853-7; Baral T N, Magez S, StijlemansB, Conrath K, Vanhollebeke B, Pays E, Muyldermans S, De Baetselier P.Experimental therapy of African trypanosomiasis with ananobody-conjugated human trypanolytic factor. Nat. Med. 2006 May; 12(5): 580-4; Coppieters K, Dreier T, Silence K, Haard H D, Lauwereys M,Casteels P, Beirnaert E, Jonckheere H, Wiele C V, Staelens L, Hostens J,Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosisfactor alpha VHH proteins derived from camelids show superior potencyand targeting to inflamed joints in a murine model of collagen-inducedarthritis. Arthritis Rheum. 2006 June; 54 (6): 1856-66]; multimerizationby introduction of additional amino acids sequences of interactingprotein domains, such as leucine zippers [Harbury P. B., Zhang T., KimP. S., et al. A switch between two-, three- and four-stranded coiledcoils in GCN4 leucine zipper mutants. Science, 1993, 262:1401-1407;Shirashi T., Suzuyama k., Okamoto H. et al. Increased cytotoxicity ofsoluble Fas ligand by fusing isoleucine zipper motif. Biochem. Biophys.Res. Communic. 2004, 322: 197-202; Chenchik A., Gudkov A., Komarov A.,Natarajan V. Reagents and methods for producing bioactive secretedpeptides. 2010. US Patent Application 20100305002], or small proteinssequences, making stable complexes [Deyev S M, Waibel R, Lebedenko E N,Schubiger A P, Plückthun A. Design of multivalent complexes using thebarnase*barstar module. Nat Biotechnol. 2003, 21(12):1486-92.].

It has also been shown [Vincke C., Loris R., Saerens D., et al. // J.Biol. Chem. 2009. V. 284. No. 5. P. 3273-3284], that these camelnanoantibodies can be “humanized” without evident loss of their specificactivity, making little number of point amino acid replacements. Thisgives potential to wide usage of nanoantibodies for passive immunizationto prevent development of various infectious diseases [Wesolowski J.,Alzogaray V., Reyelt J. et al. Single domain antibodies: promisingexperimental and therapeutic tools in infection and immunity. Med.Microbiol. Immunol. 2009; 198, 157-174.].

Method for isolation of nanoantibodies binding antigens of C.trachomatis is performed based on selection by phage display method,genetic engineering modifications encoding sequences of these antibodiesand using them as an active ingredient producer (antibody) of E. coli.Nanoantibodies (molecular weight about 12-15 kDa) are 10 times smallerthan the size of traditional classical antibodies and are fullyfunctional antigen-recognizing units, possessing numerous positivefeatures of practical importance. Nanoantibody is a easily solublesingle-domain protein with high stability (in a wide range oftemperatures and pH). This helps to avoid problems with solubility andright folding of antibodies molecules during production by prokaryoticcells and leads to significant reduction of production costs comparingto traditional methods of therapeutic monoclonal antibodies isolation ineukaryotic expression systems. Process of preservation and transport ofantibodies is significantly simplified comparing to evidently lessstable traditional antibodies. Due to their little size nanoantibodiesare characterized by better ability to penetrate in tissues. Finally,nanoantibodies facilitate genetic engineering manipulations with thepurpose of subsequent production of bispecific nanoantibodies orchimeras, consisting of the second protein with desired properties apartfrom nanoantibody.

Despite the fact that authors used production of antibodies using E.coli for illustration of this invention, it is obvious for an averageskilled specialist in this art that the volume of the present inventionalso involves other variants of realization systems for this invention,not specifically stated herein. For example, yeast cultures or othersystems standard for this art may be used as eukaryotic producer.

The method for isolation of nanoantibodies has been described inexamples 1 and 2. Choosing realization methods, with the purpose ofisolation of nanoantibodies with the selected properties fromprokaryotic expression system in accordance with one of the desiredoptions for performance of this invention, is conditioned upon thefollowing factors:

1) High expression rate, cost efficient development in large volume,provided by nanoantibodies expression in E. coli periplasm amounting to1-10 mg of 1 L of culture.

2) Simplicity of all possible genetic engineering manipulations,adaptations for specific issues, possibility to create multivalent andmultifunctional derivatives.

3) High economic efficiency of production.

The authors of this invention assumed that, as the average skilledspecialist in this art knows, primary, initial sequences ofnanoantibodies can be later adjusted or “formated” in various ways forsubsequent practical use. Thus, nanoantibodies can be initialmodules-units of more complicated multimodule products. Unification inone multivalent derivative of two, three and more monovalent primarynanoantibodies is possible. These nanoantibodies unified into oneconstruct can bind both with the same epitope of target antigen, and itsdifferent epitopes, or even with various target antigens. Combinedunification into one construct of nanoantibodies and other molecules ordrugs with isolation of multifunctional preparations, multimerization bymeans of introduction of new additional amino acid sequences,interactive protein domains such as leucine zippers or small proteinssequences, forming stable complexes, are possible. For modulation ofproperties of nanoantibody products (e.g. life time increment orpurification method enhancement) additional amino acid sequences may beintroduced into content of the end compound. It will be evident for anaverage specialist of this art that such modifications and othervariants of antibodies, underlying in this invention, are involved intovolume of this invention, because they are structural and functionalvariants of nanoantibodies. In this wise, under the term“nanoantibodies” authors of this invention mean both primary, initial“minimal” amino acid sequences of nanoantibodies, and theirmodifications obtained as a result of the designated adaptations or“formatting” and their variants. The term “antibody variant” for thepurpose of this invention means polypeptide with changes in amino acidsequence, i.e. deletion, insertion, addition or substitution of aminoacids, provided that the needed protein activity level is preserved, forinstance, at least 10% of activity of the initial nanoantibody. Severalchanges in protein variant depend on location or type of amino acidresidue in 3D protein structure. Number of changes may amount, forexample, from 1 to 30, more preferably from 1 to 15, and the mostpreferably from 1 to 5 changes in initial nanoantibody sequence. Thesechanges can take place in regions of polypeptide that are not criticalfor its function. This can be possible owing to that fact, that someamino acids possess high homology with each other and that is whytertiary structure or protein activity are not violated in this change.That is why protein characterized by homology not less than 70%,preferably, not less than 80%, more preferably, not less than 90%, andthe most preferably, not less than 95% regarding amino acid sequence ofinitial nanoantibody can be an option, provided that polypeptideactivity is preserved. Homology between amino acid sequences can beestablished using well known methods, for example, sequence alignment inBLAST 2.0 computer application, calculating three parameters: count,identity and similarity.

Deletion, insertion, addition or substitution of one or several aminoacid residues shall represent conservative mutation or conservativemutations, provided that in this case protein activity is preserved.Conservative substitution is an example of conservative mutation (s).“Conservative amino acid substitution” is a substitution when amino acidresidue is replaced by amino acid residue, having similar side chain. Inthis art amino acid families with similar side chains are determined.These families include amino acids with main side chains (for example,lysine, arginine, histidine), acidic side chains (for example, asparticacid, glutamic acid), chargeless polar side chains (for example,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (for example, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (for example, threonine, valine, isoleucine) and aromatic sidechains (for example, tyrosine, phenylalanine, tryptophan, histidine). Asfar as hypervariable regions of nanoantibodies determine their specificinteraction with antigen, that is why homological amino acidsubstitutions in these regions exactly may lead to isolation of severalsequence-varied nanoantibodies, having identical or similar properties.So an average skilled specialist of this art shall obviously understandthat the volume of this invention shall involve not only specified inthe supplement nanoantibodies sequences, but also those that can beisolated by amino acid substitutions in hypervariable regions, listed inthe sequences list as CDR to others, but with very similar properties,amino acids of conservative substitutions.

DNA fragments, that are virtually encoding the same functionalpolypeptide, can be isolated, for example, by modification of DNAfragment nucleotide sequence, encoding initial nanoantibody, forexample, by site-directed mutagenesis method, so that one or severalamino acid residues in the specific site will be deleted, substituted,inserted or added. DNA fragments, modified as described above, can beisolated by traditional treatment methods for mutation.

DNA fragments, virtually encoding the same functional polypeptide of theinitial nanoantibody, can be isolated by expression of DNA fragmentswith above mutation, and establishment of activity of the expressedproduct.

Substitution, deletion, insertion or addition of the above nucleotidesalso involve mutations occurring in nature and, for example, can beattributed to variability.

Polypeptides of nanoantibodies considered in this invention can beencoded by numerous molecules of nucleic acids, which is a result offamous in this art phenomenon of code degeneracy. The essence ofphenomenon consists in that fact that any amino acid (excludingtryptophan and methionine) in the composition of natural peptides, canbe encoded by more than one triplet nucleotide codon. Any of thesedegenerated encoding molecules of nucleic acids can be a part ofcassettes, expressing antibodies stated in accordance with thisinvention and involved into the volume of this invention.

Isolation of functional active nanoantibodies, recognizing antigens ofC. trachomatis is illustrated in examples 3, 4.

Inhibiting effect of therapeutically efficient number of nanoantibodieson the development of Chlamydia infection is illustrated in example 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of microimmunofluorescence (MIF) assaydemonstrating specific binding of C. trachomatis by aCt1 and aCt2nanoantibodies. Detection of bound nanoantibodies was performed usinganti-HA antibodies of mice and secondary antibodies to immunoglobulinsof mice, conjugated with fluorescein isothiocyanate (FITC). Specificbinding is characterized with bright-green light. X 1500, where,

A—photomicrography of MIF results with aCt1 (10 μ/ml)

—photomicrography of MIF results with aCt2 (10 μ/ml)

C. trachomatis, 2—ovalbumin protein, 3—C. pneumoniae, 4—C. muridarum,5—C. psittaci.

FIG. 2 represents results of analysis for specific binding of singledomain nanoantibodies specifically with C. trachomatis, formingintracellular inclusions in eukaryotic cells, where

McCoy cells, infected with C. trachomatis and dyed with aCt1,

nanoantibodies—noninfected McCoy cells dyed with aCt1 nanoantibodies

3—McCoy cells, infected with C. trachomatis and dyed with aCt2nanoantibodies

4—noninfected McCoy cells dyed with aCt2 nanoantibodies

FIG. 3 illustrates results of inhibiting effect analysis of aCt1 andaCt2 nanoanibodies on C. trachomatis in vitro in neutralization reactionof intracellular forms—elementary bodies of Chlamydia.

1—McCoy cells 48 hours after infection with C. trachomatis (control)

2—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 1 μg/ml aCt1

3—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 5 μg/ml aCt1

4—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 10 μg/ml aCt1

5—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 1 μg/ml aCt2

6—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 5 μg/ml aCt2

7—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 10 μg/ml aCt2.

DETAILED DESCRIPTION OF THE INVENTION

Further are represented nucleic and amino acid sequences of two selectednanoantibodies, aCt1 and aCt2, isolated concurrently by the same methodand having the desired properties: ability to specifically bindChlamydia trachomatis. Nucleic cDNA sequences encoding two selectednanoantibodies have been determined for aCt1—SEQ ID NO: 1; for aCt2—SEQID NO: 3, the relevant amino acid sequences of selected nanoantibodieshave been selected out of them: for aCt1—SEQ ID NO: 2; for aCt2—SEQ IDNO: 4. Hypervariable regions of antigen recognizing sequences ofselected nanoantibodies from left to right, from N- to C-end CDR1, CDR2and CDR3 have been underlined in the designated sequences. The arrowsshow positions of amino acid residues that are characteristic forvariable domains of specific single chain antibodies differing fromresidues in variable domains of heavy chains in classical antibodies.

EXPERIMENTAL Example 1

Isolation of library of variable domains in single chain antibodies

Immunization.

Camelus bactrianus had been consistently immunized for 5 times bysubcutaneous injection of antigenic material, mixed with equal portionof incomplete Freund's adjuvant. The product of native purifiedelementary particles of Bu-434 C. trachomatis strain (whole bacterialcells), inactivated by UV-irradiation, and protein complex product ofthe outer membrane of cell wall of Bu-434 C. trachomatis strain withoutLPS was used as an antigen. The second immunization was performed in 3weeks after the first one, followed by two more immunizations in twoweek interval. Blood (150 ml) was sampled 6 days after the lastinjection. To prevent from coagulation of the sampled blood 50 ml ofstandard phosphate buffer solution (PBS), containing heparin (100 un/ml)and EDTA (3 mM) has been added.

The blood has been twice diluted in PBS containing 1 mM EDTA. 35 ml ofdiluted blood solution was coated on surface of special medium(Histopaque-1077, Sigma) with density 1.077 g/ml and volume of 15 ml andthen was centrifuged for 20 mM at 800 g. Mononuclear cells (lymphocytesand monocytes) were sampled from interphase zone plasma/Histopaque,followed by washing with PBS, containing 1 mM EDTA.

Total RNA from B-lymphocytes was isolated using TRIzol (Invitrogen)reagent. Later at the column with oligo(dT)-cellulose poly(A)containingRNA has been purified from total RNA. RNA concentration has beendetermined using Biophotometer (Eppendorf) and the quality of isolatedRNA was verified by electrophoresis in 1.5% agarose gel withformaldehyde.

Reverse transcription reaction was performed according to standardprotocol [Sambrook et al., 1989] using reverse transcriptase H-M-MuLVand oligo(dT)15primer.

Reverse transcriptase products were used as a matrix in two stagepolymerase chain reaction and the isolated amplification products werecloned at sites NcoI(PstI) and NotI into phagemid vector, as describedabove [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens etal., 2004; Rothbauer et al., 2006]. Selection was performed similarly tothose in specified works. It was based on phage display method, wherebacteriophage M13KO7 (New England Biolabs, USA) is used as a helperphage.

Example 2

Selection of nanoantibodies specifically recognizing C. trachomatis.

Nanoantibodies were selected by phage display method using 2 products:purified elementary bodies C. trachomatis Bu-434 and protein complex ofcell wall outer membrane of Bu-434 C. trachomatis strain without LPSimmobilized at the bottom of wells of 96-well reaction plate. Highsorption polystyrene immunological plates MICROLON 600 (Greiner Bio-One)were used. One percent BSA (Sigma-Aldrich, USA) and/or 1% nonfat milk(Bio-Rad, USA) in PBS were used as blocking buffer. Process of selectionand subsequent amplification of selected phage particles (containingsingle domain nanoantibody gene inside, and expressed single domainnanoantibody forming surface phage protein pIII) was repeated, as arule, three times in series. All the manipulations were performed asdescribed in publications of Tillib S. V., Ivanova T. I., Vasiliev L. A.2010. Fingerprint analysis of nanoantibodies selection by phage displaymethod using two variants of helper phages. Acta Naturae 2010; 2 (3):100-108; Hamers-Casterman C., Atarhouch T., Muyldermans S. et al. Nature1993; 363: 446-448; Nguyen V. K., Desmyter A., Muyldermans S. Adv.Immunol. 2001; 79: 261-296; Saerens D., Kinne J., Bosmans E., WerneryU., Muyldermans S., Conrath K. J Biol Chem. 2004; 279: 51965-51972;Rothbauer U., Zolghadr K., Tillib S., et al. Nature Methods 2006; 3:887-889].

Clones sequences of selected nanoantibodies were grouped according tosimilarity of their fingerprints, obtained during electrophoreticseparation products of hydrolysis of amplified sequences of singledomain nanoantibodies concurrently with three frequent-cutterrestriction enzymes (HinfI, MspI, RsaI). cDNA sequences ofnanoantibodies (SEQ ID NO: 1 and 3) were determined (FIG. 1).Hypervariable regions CDR1, CDR2 and CDR3 of antigen recognizingsequences of selected nanoantibodies (from left to right) wereunderlined in the designated sequences.

Nanoantibodies Production

cDNA sequences of selected nanoantibodies were recloned into expressionplasmid vector—modified pHEN6 vector [Conrath K E, Lauwereys M, GalleniM, Matagne A, Frere J M, Kinne J, Wyns L, Muyldermans S. Beta-lactamaseinhibitors derived from single-domain antibody fragments elicited in theCamelidae. Antimicrob Agents Chemother. 2001; 45:2807-12] allowingattachment to C-end of (His)6-epitope nanoantibody (right afterHA-epitope, encoded in pHEN6 vector). Owing to presence of signalpeptide at the N-end of expressed sequence (pelB), developed recombinantprotein (nanoantibody) accumulated in bacterial periplasm, facilitatingefficient isolation by osmotic shock method without destruction ofrespective bacterial cells. Production of single domain nanoantibodieswas performed in E. coli (BL21 strain). Expression was induced by adding1 mM of indolyl-beta-D-galactopyranoside and the cells were incubatedwith vortexing during 7 hours at t=37oC or at night at t=29oC.Nanoantibody was isolated from periplasmatic extract using affinitychromatography in Ni-NTA-agarose using QIAExpressionist (QIAGEN, USA)purification system.

Demonstration of specificity of binding aCt1 and aCt2 nanoantibodieswith C. trachomatis.

Ability of single domain nanoantibodies to specifically bind C.trachomatis antigens was tested using MIF assay with immobilized C.trachomatis, C. pneumoniae, C. muridarum, C. psittaci antigens understandard protocol (K. Persson, J. Boman. Comparison of Five SerologicTests for Diagnosis of Acute Infections by Chlamydia pneumoniae. Clin.Diagn. Lab. Immunology, 2000, Vol. 7, No. 5, p. 739-740). (SUPPLEMENT 2)Wells with immobilized chicken ovalbumin protein were used as negativecontrol (nonspecific protein). Detection of bound aCt1 H aCt2 wasperformed using murine anti-HA antibodies and secondary antibodies tomurine immunoglobulins conjugated with fluorescein isothiocyanate(FITC). The results were evaluated by luminescent microscope with 1500×zoom. In case of specific antibodies binding with antigen bright-greenlight was observed.

FIG. 1 represents results, proving that nanoantibodies are specificallybound with immobilized C. trachomatis antigens, but not with C.pneumoniae, C. muridarum, C. psittaci.

A—photomicrography of MIF results with aCt1 (10 μ/ml)

—photomicrography of MIF results with aCt2 (10 μ/ml)

C. trachomatis, 2—ovalbumin protein, 3—C. pneumoniae, 4—C. muridarum,5—C. psittaci.

In case of immobilized C. trachomatis antigen on fragments of Figure A1and

1, bright-green light typical for FITC is observed, that is the evidenceof positive reaction of aCt1 and aCt2 nanoantibodies binding with C.trachomatis cells. In case of other immobilized antigens (A3, A4, A5),negative control (A2) for aCt1 nanoantibodies, and also antigens (

3,

4,

5), negative control (

2) for nanoantibodies aCt2 the light is not observed. This is theevidence of binding failure of the selected nanoantibodies with antigensof Chlamydia of other species and failure of nonspecific binding withovalbumin protein.

Example 4

Illustration of nanoantibodies binding aCt1 and aCt2 with eukaryoticcells, infected with C. trachomatis in vitro.

Eukaryotic culture of McCoy cells was infected with C. trachomatis underthe standard method (Bashmakov Y K, Zigangirova N A, Pashko Y P,Kapotina L N, Petyaev I M. Chlamydia trachomatis growth inhibition andrestoration of LDL-receptor level in HepG2 cells treated withmevastatin. Comp Hepatol., 2010, 28; 9:4). (SCHEDULE 3). Daily monolayerof cells was infected with C. trachomatis Bu-434 strain by applicationof C. trachomatis into cultural medium with subsequent centrifuging.Cells were incubated at 37° C. during 48 hours. Then the cells werefixed with acetone. Binding ability of aCt1 and aCt2 nanoantibodies withC. trachomatis, forming intracellular inclusions in eukaryotic cells,were tested by MIF assay under the standard protocol. Fixed uninfectedcells were used as a control. Detection of bound aCt1 and aCt2nanoantibodies was performed using murine anti-HA antibodies andsecondary antibodies to murine immunoglobulins conjugated withfluorescein isothiocyanate (FITC). In case of specific binding ofantibodies with Chlamydia in infected eukaryotic cells vacuoles, calledinclusions containing Chlamydia, bright-green light was observed.

FIG. 2 represents results proving that single domain nanoantibodiesspecifically bind to C. trachomatis, forming intracellular inclusions ineukaryotic cells, and do not bind to uninfected cells.

McCoy cells, infected with C. trachomatis and dyed with aCt1nanoantibodies—noninfected McCoy cells dyed with aCt1 nanoantibodies

3—McCoy cells, infected with C. trachomatis and dyed with aCt2nanoantibodies

4—noninfected McCoy cells dyed with aCt2 nanoantibodies

Chlamydial inclusions inside of infected cells with bright-green lighton fragments 1 and 3 are observed. There is no light in fragments 2 and4 in uninfected cells. This proves specific binding of aCt1 and aCt2nanoantibodies with C. trachomatis in cytoplasm of infected cells.

Thus, pharmaceutical products claimed in accordance with this inventionproved their applicability for detection of C. trachomatis in vitro.

Example 5

Illustration of efficiency of inhibiting action of aCt1 and aCt2nanoantibodies for development of Chlamydia infection.

To demonstrate inhibiting effect of nanoantibodies on development ofChlamydia infection in cell cultures, neutralizing effect ofnanoantibodies on extracellular forms of C. trachomatis (elementarybodies) was evaluated. For this, nanoantibodies were diluted in PBS. C.trachomatis (105 CFU) were added to nanoantibodies dilutions andincubated at 37° C. for 45 minutes. Then preincubated elementary bodieswere added to monolayer of McCoy cells, the cells were centrifuged for 1hour at 1500 rpm and incubated for 48 hours in DMEM medium withcycloheximide (1 mg/ml). The cells were fixed with acetone, then dyedwith monoclonal antibodies to MOMP protein of C. trachomatis conjugatedwith FITC adding Evans blue dye. The products were viewed byluminescence microscope under 1500× zoom. Chlamydial intracellularinclusions were detected as vacuoles with bright-green light at the redcells background.

FIG. 3 illustrates results of inhibiting effect analysis of aCt1 H aCt2nanoanibodies on C. trachomatis in vitro in neutralization reaction ofintracellular forms—elementary bodies of Chlamydia.

1—McCoy cells 48 hours after infection with C. trachomatis (control)

2—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 1 μg/ml aCt1

3—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 5 μg/ml aCt1

4—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 10 μg/ml aCt1

5—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 1 μg/ml aCt2

6—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 5 μg/ml aCt2

7—McCoy cells 48 hours after infection with C. trachomatis, preincubatedwith 10 μg/ml aCt2

Large intracellular Chlamydial inclusions with bright-green light weredetected in control substance in cells 48 hours after getting infectedby C. trachomatis. In case of preincubation of elementary bodies C.trachomatis with aCt1 and aCt2 nanoantibodies, with nanoantibodiesconcentration 5 and 10 μg/ml, significant reduction of inclusions volumein cells monolayer, and also reduction of their size comparing tocontrol were observed.

Thus, inhibiting action of aCt1 and aCt2 nanoantibodies on C.trachomatis intracellular development in vitro was illustrated.

Thus the examples with illustrations set forth prove performance ofobjective of this invention, i.e. isolation of new antibodies able toeffectively bind antigens of C. trachomatis and inhibit development ofChlamydia infection. These new antibodies have the following advantagesregarding prototype (classic monoclonal antibody): isolation, productionand storage is more cost-efficient, effective and relatively simple;their size is significantly smaller, then in classic antibodies; theypossess new structural features, allowing in principle to recognize some

hidden

for ordinary antibodies epitopes; properties of their compact structuremust lead to relative simplicity of all the possible manipulations,adjustment to specific objectives, possibilities to create on theirbasis of various multivalent and multifunctional derivatives.

What is claimed is:
 1. A nanoantibody specifically binding to a surfaceantigen of Chlamydia trachomatis, the nanoantibody comprising an aminoacid sequence SEQ ID NO:
 2. 2. A nanoantibody specifically binding to asurface antigen of Chlamydia trachomatis, the nanoantibody comprising anamino acid sequence SEQ ID NO:
 4. 3. The nanoantibody according to claim1, wherein the nanoantibody inhibits a Chlamydia infection caused by C.trachomatis.
 4. The nanoantibody according to claim 2, wherein thenanoantibody inhibits a Chlamydia infection caused by C. trachomatis. 5.A method of in vitro inhibiting a Chlamydia infection caused by C.trachomatis, the method comprising: pretreating elementary bodies of C.trachomatis by a therapeutically efficient amount of a nanoantibodyspecifically binding to a surface antigen of Chlamydia trachomatis, thenanoantibody comprising an amino acid sequence SEQ ID NO: 2; and addingthe elementary bodies of C. trachomatis to target cells being infected.6. A method of in vitro inhibiting a Chlamydia infection caused by C.trachomatis, the method comprising: pretreating elementary bodies of C.trachomatis by a therapeutically efficient amount of a nanoantibodyspecifically binding to a surface antigen of Chlamydia trachomatis, thenanoantibody comprising an amino acid sequence SEQ ID NO: 4; and addingthe elementary bodies of C. trachomatis to target cells being infected.